The chemokine CXCL12 and its G protein-coupled receptor (GPCR), CXCR4, regulate cell migration during development, immune surveillance and inflammation in normal physiology. They are also notorious for their roles in disease, particularly cancer. Recently, the atypical chemokine receptor, ACKR3, was identified as a second receptor for CXCL12 that does not signal through G proteins but instead couples to ?-arrestin. Like CXCR4, ACKR3 is expressed during development and up-regulated in cancer. Despite their medical importance, the mechanisms by which CXCR4 and ACKR3 are activated to elicit distinct functional responses are poorly understood. Biophysical, computational and mutagenesis studies have shown that CXCR4 and ACKR3 recognize CXCL12 in a structurally similar manner. However, activation of CXCR4 is sensitive to even single point mutations of the chemokine and the receptor-binding pocket, whereas virtually all ligands tested activate ACKR3. Thus, CXCR4 and ACKR3 appear to function by different mechanisms. We hypothesize that CXCR4 activation involves a precise network of residues that stabilize the active conformation of the receptor, whereas ACKR3 activation occurs by a ?wedge-like? mechanism, such that whenever any ligand docks in the receptor-binding pocket, it activates by destabilizing the inactive conformation. We propose to use single- molecule fluorescence (SMF) spectroscopy to explore the conformational dynamics and different activation mechanisms of these two receptors. We will also investigate how ligands and effectors (G proteins and ?- arrestin) control the conformations of CXCR4 and ACKR3 and thus their signaling responses. The underlying hypothesis is that GPCRs and ACRs are intrinsically dynamic, sampling multiple conformations, and that ligands and effectors mutually regulate each other to influence the receptor conformation and signaling output. Strong preliminary data support this hypothesis. Our central hypothesis will be pursued with three specific aims. 1: Establish SMF methods to monitor the conformational dynamics of CXCR4 and ACKR3 in real-time, and probe their mechanisms of activation. 2: Investigate structural mechanisms of ACKR3 activation and the allostery between agonist binding and ?-arrestin coupling. 3: Investigate structural mechanisms of CXCR4 activation and the allostery between agonist binding and G protein coupling. The innovation of this proposal is that novel SMF methods will provide experimental information on receptor dynamics and allostery that cannot be obtained with other methods. Moreover, these approaches have never been applied to chemokine receptors and very little is known about the relationship between conformational dynamics and atypical receptor activation. The studies are significant because they will provide unique insights into the distinct activation mechanisms of two therapeutically important receptors, one that is a G protein-coupled receptor and one that is ?-arrestin-coupled. Understanding how ligands and effectors control the conformational state and signaling output of these receptors should ultimately inform drug development.